Purification and cloning of an esterase from the weed black-grass (Alopecurus myosuroides), which bioactivates aryloxyphenoxypropionate herbicides.

Abstract

Carboxyesterases which activate aryloxyphenoxypropionate (AOPP) graminicides to their bioactive herbicidal acids by hydrolysing the respective ester precursors have been identified in black-grass (Alopecurus myosuroides), a problem weed of cereal crops in Northern Europe. The dominant 40 kDa carboxyesterase was purified 1700-fold and identified as a serine hydrolase by affinity labelling with a biotinylated fluorophosphonate suicide substrate. MS-MS sequencing of a peptide digest identified it to be a member of the GDSL family of serine hydrolases. The full-length A. myosuroides hydrolase (Amgdsh1) was cloned by RACE-PCR and expressed in the yeast Pichia pastoris as a secreted enzyme. Expression was associated with activity towards AOPP esters. AmGDSH1 was predicted to be glycosylated and exported to the apoplast in planta. Based on the analysis of related sequences in monocotyledonous plants an alternative classification of the GDSL plant hydrolase superfamily is suggested and their importance in endogenous metabolism and herbicide bioactivation in crops and weeds discussed.